Development of effective vaccines and immunotherapies for the treatment of infections related to biodefense will require induction of immune responses capable of eradicating the reservoirs of infection in infected hosts. For many Category A-C pathogens, this will require the stimulation of cellular immune responses capable of eliminating infected cells. Delivery of antigen and adjuvants in a manner that induces effective, antigen-specific cellular immune responses is a critical challenge for the design of vaccines and immunotherapies for biodefense. Effective immunization strategies will require 1) an antigen delivery strategy capable of delivering antigen to dendritic cells to enable class I and class II restricted processing and presentation, and 2) adjuvant strategies designed to engineer or "condition" DCs to express the desired antigen presentation function. The skin has long been recognized as a preferred site for vaccination. With numerous readily accessible resident dendritic cells (DCs), and accessory cells that have the capacity to secrete inflammatory mediators, the skin constitutes an ideal microenvironment to induce and control antigen-specific immune responses. Several cutaneous antigen delivery technologies are under development and show considerable promise, including protein-based and genetic immunization strategies. On the other hand, a major obstacle to the development of cutaneous immunization strategies is the observed predominance of Th2 skewed immune responses, particularly in response to genetic immunization. In Specific Aim 1, we propose to evaluate the potential of imidazoquinolinamines as topically applied Th-1 skewing cutaneous adjuvants for use in vaccines and immunotherapies for biodefense. This class of synthetic small molecules has well-documented effects on innate and acquired immune responses. They are currently the only clinically approved topical agents capable of inducing Th-1 mediated immune responses, in contrast to the many Th-2 inducing agents currently approved or in development. A second major obstacle for the development of cutaneous immunization strategies has been the lack of an appropriate model to evaluate the mechanisms of immunogenicity and effectiveness in human skin. Currently, cutaneous immunization strategies are evaluated primarily through murine or non-human primate systems. There are considerable differences between human vs. murine or primate skin with regard to architecture and composition, which underlie observed functional differences in immune activation, transfection, and transgene expression. Consequently, results obtained in murine or primate models have not necessarily translated to the human cutaneous microenvironment. In Specific Aim 2, we propose to engineer human skin to enable generation of Th1 skewed immune responses by translating our hypothesis from Specific Aim 1 directly to human skin. Further, in Specific Aim 3, we propose to use the established human skin platform to comparatively evaluate cutaneous adjuvants that demonstrate high potential for use in biodefense, as determined in cooperation with NIAID Program Staff. Together, these studies are designed to demonstrate feasibility and provide mechanistic and preclinical rationale for continuing development of cutaneous immunization strategies, including validation of a preclinical human skin model for the comparative evaluation of cutaneous immunization strategies for clinical applications.